Rotaviruses (RV) are the single most important cause of severe diarrhea in infants and young children worldwide. These viruses are also common causes of disease in healthy adults, the elderly and the immunocompromised. Virtually all mammalian species are efficiently infected with their own homologous host- species RV but not by heterologous RV. Recently, effective, live attenuated RV vaccines became commercially available, but their mechanisms of action and molecular basis for attenuation are not understood. Infection with RV is predominantly restricted to the villous epithelium of the small intestine. Of note, the basis for host-range restriction of heterologous RV replication in intestinal cells remains unknown. Due to their relatively small genomic size, host-range and tissue tropism, rotaviruses are a highly tractable model system for study of the interaction between host epithelium and enteric pathogens. RVs are also potential vectors for targeting the human small bowel epithelium and an ideal system for assessing vaccination strategies against enteric pathogens. It is our objective to continue studies of RV-host interactions with the goal of better understanding enteric microbial pathogenesis. The specific aims of the proposal are to: 1) Characterize the genetic basis for host-restricted intestinal cell tropism of selected homologous and heterologous RVs using viral reassortants in a murine model. It is our hypothesis that one of the RV surface proteins (VP4), which mediates cell entry and the RV IRF3 antagonist protein, NSP1, which blocks the host interferon response, are responsible for determining the phenotype of host restricted intestinal replication. 2) Characterize early events in RV cell entry in an in vitro model of polarized epithelium and determine the conformations of VP4 and VP7 that mediate cell entry. It is not clear how the large, non-enveloped icosahedral RV particle enters the cytoplasm of a polarized enterocyte. We will use a combination of highly specific monoclonal antibodies, confocal microscopy and cell biologic techniques to illuminate this process. Cell entry events contribute to the varying intestinal cell tropisms of different homologous and heterologous RV strains. We hypothesize that the entry process is characterized by critical changes in the structure and cellular localization of the two RV surface proteins. 3) Develop a tractable reverse-genetics system to manipulate the genome of a replication competent rotavirus. In order to take RV pathogenesis and cell entry studies to the next level, methodologies to directly modify the RV double-stranded, segmented RNA genome are needed. Such a system will also potentially enable us and other investigators to utilize the exquisite entero-tropism of RVs to design unique targeting vectors for other infectious diseases. It is our hypothesis that we can accomplish this aim by utilizing approaches recently validated for reovirus and orbiviruses.